The QIAGEN PCR Cloningplus Kit is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.

Transformation without recovery incubation.

QIAGEN EZ Competent Cells do not require recovery incubation. QIAGEN EZ Competent Cells (>108 cfu/µg DNA), TOP 10F (Supplier I; >109 cfu/µg DNA), and JM109 (Supplier P; >108 cfu/µg DNA) competent cells were transformed with pUC18 plasmid DNA. The recommended protocol from each supplier was followed, except that all cells were plated immediately onto agar/ampicillin plates without a recovery incubation in SOC medium. Colony numbers were converted to relative percentages, with QIAGEN EZ Competent Cells set at 100%. Colony numbers were not normalized for transformation efficiency. Normalization would result in an even higher transformation efficiency for QIAGEN EZ Competent Cells.

Performance

The QIAGEN PCR Cloningplus Kit combines the latest ligation technology with a unique combination of time-saving features for fast, easy, and highly efficient cloning of PCR products generated using Taq and other nonproofreading DNA polymerases. The QIAGEN PCR Cloningplus Kit outperformed kits tested from other suppliers, ensuring successful results. Cloning into the pDrive Cloning Vector is much faster compared with TA-based cloning vectors (see figures "Highly specific cloning with a shorter ligation time of 30 min" and Highly specific cloning with a ligation time of 4 h", and table). The QIAGEN PCR Cloningplus Kit is supplied with QIAGEN EZ Competent Cells, which do not require this time-consuming recovery incubation to achieve high-efficiency transformation (>108 colony forming units (CFU) per microgram DNA; see figure "Transformation without recovery incubation").

Time from PCR product to plated cells for different cloning methods

QIAGEN PCR Cloningplus Kit

Topoisomerase-mediated cloning kit

TA-based cloning kit

Conventional ligase cloning

40 min

≥70 min

≥5.5 h

≥7.5 h

Principle

The pDrive Cloning Vector (see figure "pDrive Cloning Vector") provides highly efficient cloning of PCR products through UA hybridization. The vector is supplied in a linear form with a U overhang at each 3' end, which hybridizes with high specificity to the A overhang of PCR products generated by Taq and other nonproofreading DNA polymerases. The pDrive Cloning Vector has a number of useful features designed to facilitate analysis of cloned PCR products. These include a large number of unique restriction enzyme recognition sites, universal sequencing primer sites, and promoters for in vitro transcription. In addition, the vector allows both ampicillin and kanamycin selection, as well as blue/white screening of recombinant colonies.

PCR products are efficiently cloned into the pDrive Cloning Vector in less time than is required for TA-based cloning vectors (see figures "Highly specific cloning with a shorter ligation time of 30 min" and Highly specific cloning with a ligation tme of 4 h"). Furthermore, as T is the most likely base to hybridize to noncomplementary bases (i.e., G, C, and T), vectors with a T overhang are more likely to self-anneal or to clone primers or annealed primers, leading to an increased number of false-positive colonies. In contrast, the higher cloning efficiency of the pDrive Cloning Vector indicates that U has a lower tolerance for nonspecific base pairing.

The Ligation Master Mix, supplied in a convenient premixed format, is specifically designed to provide optimal hybridization conditions for efficient cloning.

The use of QIAGEN EZ Competent Cells makes the cloning procedure even faster and more convenient. Transformed cells are usually incubated in SOC medium to recover and to have time to express antibiotic resistance. In contrast, QIAGEN EZ Competent Cells do not require this time-consuming recovery incubation to achieve high-efficiency transformation (>108 colony forming units (CFU) per microgram DNA; see figure "Transformation without recovery incubation").

Components included with the QIAGEN PCR Cloningplus Kit

Component

Included

Concentration

pDrive Cloning Vector

50 ng/µl

Ligation Master Mix

2x solution

Distilled Water

–

QIAGEN EZ Component Cells

–

SOC medium

1x solution

Procedure

Simply mix the PCR product directly with pDrive Cloning Vector and Ligation Master Mix, incubate, and then add the ligation reaction to competent cells for transformation. QIAGEN EZ Competent Cells do not require a recovery incubation in SOC medium after transformation, and therefore can be plated immediately onto agar/ampicillin plates.

The QIAGEN PCR Cloningplus Kit is suitable for cloning of any PCR product that has a single A overhang at each 3' end. PCR products generated usingTaqand other nonproofreading DNA polymerases can be directly cloned without any preparation. Kits offering proofreading DNA polymerases, such as the HotStar HiFidelity Polymerase Kit and the QIAGEN LongRange PCR Kit, generate PCR products with A overhangs, and are also highly suited for direct use with the QIAGEN PCR Cloningplus Kit.